Polypeptide and gene encoding the same

ABSTRACT

A polypeptide having the amino acid sequence as set forth in SEQ ID NO: 2, 4, or 6, a DNA encoding the same, and an antibody against said polypeptide, and the use thereof. The above amino acid sequence has a homology with chondromodulin-I that has an effect of controlling the growth and differentiation of chondrocytes and inhibiting angiogenesis.

This is a divisional of U.S. Ser. No. 11/055,967 filed Feb. 14, 2005,which is a divisional of U.S. Ser. No. 10/089,600 filed Mar. 29, 2002,now abandoned, which is a National Stage application filed under 35U.S.C. § 371 of PCT/JP00/06804 filed Sep. 29, 2000.

FIELD OF THE INVENTION

The present invention relates to a novel human, mouse and ratpolypeptide having a homology in the amino acid sequence withchondromodulin-I (ChM-I) that is known to have an effect of controllingthe growth and differentiation of chondrocytes and inhibitingangiogenesis, and a human, mouse and rat gene (hereinafter referred toas “ChM1L gene”) encoding the same.

BACKGROUND ART

Almost all the bones of mammals are formed through a mechanism called“endochondral bone formation” in which chondrocytes calcify via thegrowth and differentiation thereof, and are finally replaced with bone.It is known that a variety of hormones and growth factors participate inthis series of process, including insulin-like growth factor (IGF1,IGF2), fibroblast growth factor (FGF), transforming growth factor (TGF),growth hormone and the like. Hiraki et al. isolated ChM-I gene as afactor, in addition to the above hormones and growth factors, thatfacilitates the growth and differentiation of chondrocytes (Biochem.Biophys. Res. Commun., 175, 971-977, 1991, European Patent PublicationNo. 473080). Human ChM-I is synthesized as a type II membrane proteincomprising 334 amino acid residues and, after glycosylation, undergoesprocessing with a result that the C-terminal portion comprising 120amino acid residues are extracellularly secreted (Hiraki et al., Eur. J.Biochem. 260, 869-878, 1999). ChM-I not only promotes the growth ofcultured chondrocytes but potently promotes proteoglycan synthesis andthe colony formation of chondrocytes in agarose (Inoue et al., Biochem.Biophys. Res. Commun., 241, 395-400, 1997). ChM-I also promotes thegrowth of osteoblasts (Mori et al., FEBS Letters, 406, 310-314, 1997).

On the other hand, it has long been pointed out that cartilage remainsnot only avascular but resistant to infiltration of blood vessels.Hiraki et al. attempted to purify a growth inhibiting factor forvascular endothelial cells from the extracts of cartilaginous tissue,and have succeeded in the complete purification thereof. As a result, itwas found to be ChM-I (Hiraki et al., FEBS Letters, 415, 321-324, 1997;Hiraki et al., J. Biol. Chem., 272, 32419-32426, 1997). Generally, thecartilaginous tissue is characterized by being avascular, but in thereplacement to the bone tissue, it is believed, infiltration of bloodvessels into the cartilaginous tissue is required. In the scheduledregion of vascular invasion, the hypertrophy of cartilaginous tissue andthe calcification of cartilage matrix occur prior to vascular invasionto be ready for forming the primary point of ossification. In the regionwhere the hypertrophic cartilage and the subsequent ossified cartilageappear, the expression of ChM-I dramatically decreases. Thus, althoughthe expression of the ChM-I gene is cartilage-specific, it is limited tothe avascular cartilage that is resistant to vascular invasion. Asdescribed above, it is believed that ChM-I not only promotes the growth,differentiation, and maturing of cartilage but inhibits the infiltrationof blood vessels by inhibiting the growth of vascular endothelial cells.Thus, the expression in the avascular cartilage and the disappearance ofexpression in the ossified layer prior to vascular invasion are in goodagreement with the bifunctional effect of ChM-I.

In the cartilaginous tissue, bFGF that is a potent angiogenic factor isaccumulated in pericellular space in large quantities, and it has beenelucidated that ChM-I is present in interterritorial space in such a wayas to surround bFGF (Hiraki et al., J. Biol. Chem., 272, 32419-32426,1997). Thus, in the avascular cartilage, ChM-I is present in a form thatmasks the angiogenic factor, and it is thought that the angiogeneniceffect of ChM-I may account for the absence of blood vessels in thecartilage (Tanpakusitsu kagaku koso, Vol. 40, No. 5, 1995). It has alsobeen confirmed that ChM-I suppresses the growth of tumor cells byinhibiting the infiltration of blood vessels into human tumor cells invivo (Hayami et al., FEBS Letters, 458, 436-440, 1999). The expressionanalysis of ChM-I in various mouse tissues revealed that ChM-I isexpressed in the eye and the thymus in addition to the cartilage, butthe function of ChM-I in these tissues has yet to be elucidated(Shukunami et al., Int. J. Dev. Biol. 43, 39-49, 1999).

The growth and the expression of differentiation function ofchondrocytes plays an important role in the healing process fromfracture or various cartilage diseases. Thus ChM-I, a factor thatpromotes the growth and differentiation of chondrocytes, is a promisingcandidate for an agent that promotes the growth of chondrocytes (Kokai(Japanese Unexamined Patent Publication) No. 7-138295). In the growth ormetastasis of tumor cells, infiltration of blood vessels into tissues isrequired to obtain energy necessary therefore. Therefore ChM-I that hasan effect of inhibiting angiogenesis is also a likely candidate for ananti-cancer agent (Kokai (Japanese Unexamined Patent Publication) No.7-138295). As described above, ChM-I not only controls the growth anddifferentiation of chondrocytes but inhibits angiogenesis, and hence itsapplication into drugs is being awaited.

In recent years, biotechnology has made rapid progress, and inassociation with the development of the human genome project as well, agreat number of new genes are being cloned. It is said that the numberof human genes amounts to about 100,000, and among the genes groups ofmolecules having a homology in the amino acid sequences sometimes formfamilies. As the groups of molecules having a homology in the amino acidsequences, various gene families are known such as the TNF family, theTNF receptor family, the chemokine family, G-protein coupled receptorfamily and many other gene families. For example, as the moleculesbelonging to the TNF family, there are known about 20 moleculesincluding tumor necrosis factor α (TNFα, Pennica et al., Nature 312,724, 1984), Fas ligand (FasL, Suda et al., Cell 75, 1167, 1993),TNF-related apoptosis-inducing ligand (TRAIL, Steven et al., Immunity 3,673, 1995), B lymphocyte stimulator (BLYS, Moore et al., Science 285,260-263, 1999), and the like.

The molecules belonging to the TNF family are type II membrane proteinsand have a homology in the amino acid sequence in the extracellularregion. Although homology in the amino acid sequence is noted, thesemolecules have been demonstrated to have their inherent functions, andtheir application as pharmaceutical agents have been attempted in avariety of diseases. It has also been shown that the molecules of theTNF family have their unique receptors, and the application thereof aspharmaceutical agents have also been attempted. In fact, some have beenapproved as pharmaceutical products (for example, soluble TNF receptor,by Immunex). Research and development is also in progress on antibodiesagainst these molecules as pharmaceutical drugs, and in fact, some havebeen approved as pharmaceutical products (for example, anti-TNF-αantibody, by Centocore). As examples of molecules having a homology inthe amino acid sequence that were applied into the development ofpharmaceutical products, the TNF family and the TNF receptor family wereillustrated as above. Some of the underlying reasons that enabled theapplication of these molecules into pharmaceutical products are thefacts that the functions of each of these molecules were analyzed andthe similarity and the difference between them were elucidated.

Molecules of the TNF family have the structure of type II membraneproteins and since many of them are expressed mostly in the blood systemand the lymphatic system, they have a lot in common in terms ofexperimental techniques and samples. It is therefore expected that whena new gene belonging to the TNF family was discovered, the speed atwhich its function was analyzed must have been faster than the moleculesdiscovered earlier. Thus, the discovery of a novel gene having ahomology in the amino acid sequence and the analysis of its functionwould not only facilitates the functional analysis of novel genes to bediscovered in the future but the result of analysis permits itscomparison with the existing molecules, and therefore it is expectedthat more detailed findings on the functions of the existing moleculescould be obtained.

Generally, when a novel gene encoding a protein having a homology in theamino acid sequence with existing molecules is cloned, the techniquesand materials to be used for functional analysis may be referred to theexamples of the existing molecules. However, even a molecule having ahomology in the amino acid sequence is thought to have its own uniquefunction as in the above-mentioned TNF family, and thus when itsapplication into pharmaceutical products is envisaged, it is necessaryto demonstrate the expression and purification of the recombinantprotein, the generation of antibody, the expression of mRNA and proteinat various tissues and the like, and thereby to clarify the differencein the structure and function from the existing molecules.

DISCLOSURE OF THE INVENTION

Thus, it is an object of the present invention to provide a newpolypeptide similar to ChM-I and a gene encoding the same. It is also anobject of the present invention to implement the generation of antibodyagainst said polypeptide, the analysis of expression levels of said geneand the polypeptide, the expression and structural analysis of therecombinant protein and the like in order to clarify its similarity anddifference with ChM-I, and to elucidate the function so as to enable theelucidation of pathological states, diagnosis, treatment etc. ofdiseases in which they are involved.

ChM-I is a type II membrane protein that regulates the growth anddifferentiation of chondrocytes and inhibits angiogenesis, and is apromising candidate for application into pharmaceutical products. Thus,once a gene encoding a new polypeptide similar to ChM-I has beenprovided, it is believed, its expression level in various cells and itsstructure and function can be analyzed, and the analysis of theexpression products would enable the elucidation of pathology, diagnosisand treatment etc. of diseases in which they are involved. At present,however, there are no reports on molecules having a homology with theamino acid sequence of ChM-I, and it is unknown whether ChM-I forms agene family or not. Thus, if a new polypeptide similar to ChM-I and agene encoding the same are shown to be present, the analysis ofstructure, function etc. thereof would permit the study on itssimilarity and difference with ChM-I, which in turn would acceleratesthe elucidation of physiological functions of the molecules with oneanother, the elucidation of pathological states in which these moleculesare involved, diagnosis, the development of therapeutic agents and thelike.

After intensive and extensive research to attain the above purposes, theinventors of the present invention have succeeded in isolating a gene(ChM1L gene) that meets the above purposes, from human, mouse and ratcDNA libraries, and carried out the analysis of its expression level invarious tissues, the generation of an antibody against said polypeptide,the expression of a polypeptide encoded by said gene in a mammaliancell, its detection and purification and the like, demonstrating thatsaid polypeptide has an effect of inhibiting angiogenesis, and we herebyhave completed the present invention.

Thus, the present invention is a gene encoding a polypeptide thatsubstantially comprises the amino acid sequence as set forth in SEQ IDNO: 2, 4, and 6. As the above gene, there can be mentioned thenucleotide sequence represented by SEQ ID NO: 1, 3, and 5.

Furthermore, the present invention is a polypeptide encoded by a human,mouse and rat gene which polypeptide substantially comprises the aminoacid sequence as set forth in SEQ ID NO: 2, 4 and 6.

The present invention is also an oligonucleotide probe that hybridizesto at least part of the above gene.

The present invention is also a recombinant DNA comprising the abovegene.

The present invention is also a transformant transformed with the aboverecombinant DNA.

The present invention is also a method of producing the abovepolypeptide which method comprises culturing the above transformant andharvesting a polypeptide encoded by the gene of the present inventionfrom the culture.

The present invention is also a monoclonal antibody or polyclonalantibody that specifically reacts with the above polypeptide.

The present invention is also a hybridoma that produces the abovemonoclonal antibody that is obtained by fusing an antibody-producingcell immunized with the above polypeptide to a myeloma cell.

The present invention is also a reagent for detecting genes said reagentcomprising the above oligonucleotide probe.

The present invention is also a diagnostic kit that comprises the abovepolypeptide and the above monoclonal antibody or polyclonal antibody.

The present invention is also a pharmaceutical composition comprising apolypeptide encoded by the gene that substantially comprises the aminoacid sequence as set forth in SEQ ID NO: 2, 4, or 6.

The present invention is also a pharmaceutical composition comprising amonoclonal antibody or a polyclonal antibody that specifically reactswith the above polypeptide.

The present invention is also a pharmaceutical composition comprising anantisense oligonucleotide that specifically hybridizes to part of theabove gene.

The present invention is also a pharmaceutical composition comprising anucleic acid that can be used in gene therapy said compositioncomprising at least part of the above gene.

The present invention is also a polypeptide wherein the abovepolypeptide is a membrane-bound form.

The present invention is also a gene encoding the above membrane-boundpolypeptide.

The present invention is also a gene wherein the above human gene ispresent on chromosome X.

The present invention is also a polypeptide wherein the abovepolypeptide has an effect of inhibiting angiogenesis.

The present invention is also a gene encoding the above polypeptide thathas the above effect of inhibiting angiogenesis.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1A is a result in which the homology of amino acid sequences ofhuman ChM1L and human ChM-I were compared.

FIG. 1B is a result in which the homology of amino acid sequences ofhuman, mouse and rat ChM1L were compared.

FIG. 2 shows a hydrophobic profile of the amino acid sequences of humanChM-I, human ChM1L, and mouse ChM1L.

FIG. 3 shows the result of expression analysis of ChM1L mRNA in varioustissues of adult and fetal mice, and the expression analysis of ChM1Land ChM-I mRNA in the developmental stage of a fetal mouse.

FIG. 4 shows a result in which human and mouse ChM1L proteins wereexpressed in COS7 cells and detected by Western blot. (a) shows theresult in which Mock (lane 1), human ChM1L (lane 2), and mouse ChM1L(lane 3) were transfected, and the cellular components were subjected toelectrophoresis and stained with Coomassie brilliant blue; (c) shows theresult in which the same samples as above were detected by Western blotusing anti-ChM1L peptide antibody; (b) shows the result in which Mock(lane 1), human ChM1L (His-tagged) (lane 2), and mouse ChM1L(His-tagged) (lane 3) were transfected, and the cellular components wereelectrophoresed and stained with Coomassie brilliant blue; (d) shows theresult in which the same samples as above were detected by Western blotusing anti-His tag antibody.

FIG. 5 shows the result in which soluble ChM1L (lane 2) and Mock(lane 1) expressed in COS7 cells were detected by Western blot usinganti-FLAG M2 antibody.

FIG. 6 shows the result in which mouse ChM1L (His-tagged) protein wasexpressed in COS7 cells, and after the cellular components wererecovered they were subjected to a deglycosylation, and the ChM1Lprotein was detected by Western blot using anti-His tag antibodyfollowed by the analysis of glycosylations. Lane 1 represents the resultof Western blot of a non-treated sample, lane 2 represents that of aNANase II + O-glycosidase DS + PNGase-treated sample, lane 3 representsthat of a NANase II-treated sample, lane 4 represents that of anO-glycosidase DS-treated sample, and lane 5 represents that of aPNGase-treated sample.

FIG. 7 shows the result in which the expression of ChM1L protein inmouse rib cartilage was detected by immunostaining using anti-ChM1Lpolypeptide antibody.

FIG. 8 shows the result in which a soluble human ChM1L protein that wasexpressed in the culture liquid of COS7 cells was purified by affinitychromatography using anti-FLAG M2 affinity gel, electrophoresed, andstained with Coomassie brilliant blue. Lane 1 shows the result ofelectrophoresis of the culture supernatant of COS7 cells and lane 2shows that of the purified ChM1L protein.

FIG. 9 shows the result in which the tube-like structure-forming systemof the human umbilical vein endothelial cells were treated with (a) thebuffer alone, (b) 20 μg of bovine serum albumin (BSA), (c) 10 μg ofsoluble human ChM1L, (d) 20 μg of soluble human ChM1L, (e) 1 μg ofplatelet factor 4 (PF-4), and (f) 10 μg of PF-4.

BEST MODE FOR CARRYING OUT THE INVENTION

In accordance with the present invention, “substantially comprise” meansthat the gene or polypeptide of the present invention, as long as itretains its function, may have mutations such as substitution,insertion, or deletion in the nucleotide sequence as set forth in SEQ IDNO: 1, 3, or 5 or the amino acid sequence as set forth in SEQ ID NO: 2,4, or 6.

The ChM1L gene sequence of the present invention may be obtained by theRACE method (RACE: Rapid amplification of cDNA ends; Frohman, M. A. etal., Proc. Natl. Acad. Sci. USA, 85, 8998-9002, 1988), the outline ofwhich method is as follows:

Generally, the RACE method enables one to obtain a full-length cDNA inan efficient manner, when a portion of a cDNA sequence is known. Primersare constructed from known sequence regions to permit elongation in eachof the 3′-end or 5′-end direction, and then cDNA is amplified by thepolymerase chain reaction (PCR, Science, 230, 1350-1354, 1985). When aPCR method is carried out, primers that specifically anneal are used inthe known region, and primers that anneal to the sequence tagged by aligation reaction are used in the 3′-end and the 5′-end. Thus, theregions amplified by PCR contain unknown regions. The isolation andpurification of the amplified cDNA fragment can be performed accordingto a standard method as described below, for example gel electrophoresismay be employed. The determination of nucleotide sequence of the DNAfragment thus obtained may be performed according to a standard methodsuch as the dideoxy method (Proc. Natl. Acad. Sci. USA, 74, 5463-5467,1977) and the Maxam-Gilbert method (Methods in Enzymology, 65, 499,1980). Such determination of nucleotide sequences may also be carriedout using commercially available sequencing kits, etc.

More specifically, it is outlined as follows, though more detailedexplanations thereof will be made hereinafter in Example 2. Using theamino acid sequence of human ChM-I, TBLASTN search was performed for theEST data base (dbEST, EST: Expressed sequence tag) in the DNA data bankof Japan (DDBJ) to detect an EST file, GenbanK accession numberAI123839. AI123839, which is a nucleotide sequence fragment registeredin dbEST, was found for the first time by the above TBLASTN search to bea novel gene fragment encoding an amino acid sequence similar to ChM-I.Thus, primers were synthesized from part of the sequence of cDNAobtained from dbEST, and the sequence of human ChM1L gene was determinedusing the RACE method. Subsequently, the sequences of mouse and ratChM1L genes were similarly determined. The sequences of human, mouse andrat ChM1L genes are shown in SEQ ID NO: 1, 3 and 5, and the amino acidsequences of the peptides encoded thereby are shown in SEQ ID NO: 2, 4and 6.

The polypeptides encoded by the ChM1L genes of the present invention arecomposed of 317 amino acids (SEQ ID NO: 2, 4 and 6). The amino acidsequence of ChM1L has a homology with ChM-I, in particular a very highhomology with the C-terminal portion that is extracellularly secretedafter the processing of ChM-I (FIG. 1( a)). The amino acid sequences ofChM1L have a very high homology in between humans, mice, and rats (FIG.1( b)). From the hydrophobicity analysis of the amino acid sequence,ChM1L similarly to ChM-I is thought to be a molecule having thestructure of the type II membrane proteins (FIG. 2). As shown in FIG. 2,in both of said polypeptide and ChM-I, a hydrophobic domain comprisingabout 20 amino acids that is uniquely found in molecules having amembrane-binding activity is present in the vicinity of several dozenamino acids from the N-terminal. That said polypeptide is a moleculehaving the type II membrane protein structure was also demonstrated bythe result in Example 8 in which said polypeptide was expressed in COS7cells (FIG. 4).

The human ChM1L gene of the present invention was shown, as describedbelow in Example 12, to be present on chromosome X (GenbanK accessionNo. AL035608).

As the ChM1L gene of the present invention, there can be mentioned cDNA,chemically synthesized DNA, DNA isolated by PCR, genomic DNA andcombinations thereof. Using a standard method, said genomic DNA may alsobe isolated by hybridization with the ChM1L gene that is disclosedherein. RNA that was transcribed from said ChM1L gene is alsoencompassed in the present invention. The sequences of the gene of thepresent invention represented by SEQ ID NO: 1, 3 and 5 are a combinationexample of codons representing amino acid residues encoded by them. TheChM1L gene of the present invention is not limited to this, and it isalso possible to have a DNA sequence obtained by combining codons toamino acid residues and then selected. The selection of said codon maybe performed according to a standard method, and for example thefrequency of use of host's codons to be used is taken into account(Nucleic Acids Research, 9, 43-74, 1981).

Furthermore, the ChM1L gene of the present invention encompasses DNAsequences encoding mutants in which parts of the amino acid sequence asset forth in SEQ ID NO: 2, 4, and 6 are substituted, deleted, or added.The production, modification (mutation) and the like of thesepolypeptides may naturally occur, and can be obtained bypost-translational modification or by a genetic engineering method suchas site-specific mutagenesis (Methods in Enzymology, 154, 350, 367-382,1987; ibid., 100, 468, 1983; Nucleic Acids Research, 12, 9441, 1984;Zoku Seikagaku Jikken Koza 1 (Sequel to Biochemistry ExperimentalSeries 1) “Idensi Kenkyuuhou II (Gene Study Method II)”, edited by TheJapanese Biochemical Society, 105, 1986) and the like.

The production of the ChM1L gene of the present invention may be readilyperformed by a common genetic engineering method based on the sequenceinformation of the ChM1L gene of the present invention (MolecularCloning, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989; ZokuSeikagaku Jikken Koza (Sequel to Biochemistry Experimental Series)“Idensi Kenkyuuhou I, II, III (Gene Study Method I, II, III)”, edited byThe Japanese Biochemical Society, 1986).

This can be attained by, for example, selecting the desired clones froma cDNA library (prepared from a suitable cell source that expresses theChM1L gene according to a standard method) using suitable probes andantibodies unique to the gene of the present invention (Proc. Natl.Acad. Sci. USA, 78, 6613, 1981; Science, 222, 778, 1983, and the like).

In the above method, as the cell source, there can be illustratedvarious cells, tissues, and cultured cells derived therefrom and thelike that express the ChM1L gene, from which any of separation of totalRNA, separation and purification of mRNA, and conversion (synthesis)into cDNA, cloning etc. may be performed according to a standard method.cDNA libraries are commercially available, and in the present invention,these cDNA libraries such as various cDNA libraries commerciallyavailable from Clontech can be used.

The screening of the ChM1L gene of the present invention from a cDNAlibrary may be carried out according to the above standard method. Asthe above screening method for, for example a polypeptide produced bycDNA, there can be illustrated a method of selecting a correspondingcDNA clone by immunological screening using a specific antibody againsta polypeptide encoded by the ChM1L gene of the present invention, aplaque hybridization method using a probe that selectively binds to thenucleotide sequence of interest, a colony hybridization method and thelike as well as combinations thereof. Probes that may be used hereininclude a DNA sequence that was chemically synthesized based on theinformation on the DNA sequence of the ChM1L gene of the presentinvention, the ChM1L gene of the present invention that was alreadyobtained and fragments thereof.

In order to obtain the ChM1L gene of the present invention, a PCR methodfor amplifying DNA/RNA can be preferably used. Primes for use in such amethod can be appropriately selected based on the sequence informationof the ChM1L gene of the present invention that was elucidated for thefirst time by the present invention, and can be synthesized according toa standard method.

More specifically, it is outlined as follows, though more detailedexplanations thereof will be made hereinafter in Example 2. A primer issynthesized so as to contain the coding sequence of the ChM1L gene, andthe it is used to amplify the ChM1L gene using a PCR method. Then, it iselectrophoresed and the band of interest is excised, from which DNA ispurified. The purified DNA and the plasmid vector are ligated andtransformed into Escherichia coli (E. coli). Subsequently, the plasmidis purified from the E. coli culture liquid and the integration of thesequence of interest is confirmed using a DNA sequencer. The ChM1L genethus cloned can be transferred to other plasmid vectors or virus vectorsusing suitable restriction enzymes.

Using the ChM1L gene (cDNA and genomic DNA) thus obtained, it ispossible to create a genetically modified animal in which the expressionof the ChM1L gene is increased, decreased, or lost by a standard method.

Based on the sequence information of the ChM1L gene of the presentinvention, the expression of the ChM1L gene of the present invention invarious tissues can be detected using part or all of the nucleotidesequence of said gene. This can be advantageously accomplished accordingto standard methods such as the RT-PCR (reverse transcribed-polymerasechain reaction) (Kawasaki, E. S., et al., Amplification of RNA. In PCRProtocol, A Guide to methods and applications, Academic Press, Inc.,SanDiego, 21-27, 1989) method, Northern blotting analysis (MolecularCloning, Cold Spring Harbor Laboratory, 1989) and the like. Primers forthe RT-PCR method and probes for Northern blotting analysis are notlimited, as long as they are the sequences capable of specificallydetecting the ChM1L gene, and such sequences can be appropriatelydetermined based on the nucleotide sequence of the ChM1L gene of thepresent invention. Thus, the present invention provides primers and/orprobes useful for the detection of the ChM1L gene. The above probes mayalso be used for the detection of genomic DNA by Southern blottinganalysis.

As means to detect the expression of ChM1L mRNA, there can beillustrated the RT-PCR method described in Example 6. It is outlined asfollows, though more detailed explanations thereof will be madehereinafter in Example 6.

After extracting each tissue and extracting RNA therefrom, it issubjected to a reverse transcription reaction to synthesize cDNA. Usingthis cDNA as a template, a PCR reaction is carried out, and the reactionmixture obtained is subjected to electrophoresis on agarose gel.Examining the band under UV irradiation, the amount of the ChM1L geneexpressed in each tissue was detected. As a result, the expression ofChM1L mRNA in each tissue of an adult mouse was observed in the brain,the eyeball, the skeletal muscle, the whole rib and the thyroid (FIG. 3(a)). On the other hand, the expression of ChM-I mRNA in the mouse hasbeen confirmed in the eyeball, the thymus, the cartilage and the wholerib (Shukunami et al., J. Dev. Biol. 43, 39-49, 1999). Thus, it wasrevealed that ChM1L and ChM-I are expressed at different tissues in theliving body, and thus their physiological functions were considered tobe different. The expression of ChM1L was observed, even at tissues suchas the brain, the skeletal muscle, and the thyroid in which noexpression of ChM-I has been confirmed.

Since ChM1L is also expressed in the eyeball that is a tissue in whichChM-I expression has been observed and that is resistant to vascularinvasion and the whole rib containing cartilage, ChM1L is thought to beinvolved in angiogenesis. These results suggest that ChM1L may beassociated with brain-related diseases such as Alzheimer's disease,skeletal muscle-related diseases such as muscular dystrophy,thyroid-related diseases such as Basedow's disease, eyeball-relateddiseases such as diabetic retinopathy, cartilaginous tissue-relateddiseases such as osteoarthritis and rheumatoid arthritis, andangiogenesis-related diseases such as cancer. Thus, the ChM1L gene andthe ChM1L polypeptide of the present invention, antagonists and agoniststo ChM1L including antibody that binds to ChM1L, agents that promote orreduce the expression of the ChM1L gene, and the like are considered tobe used as therapeutic agents for these diseases. The agonists andantagonists mentioned above are intended to include peptides, proteins,low molecular weight compounds and the like, but the physical propertiesare not limited to them as long as they retain the function.

In various tissues of a fetus, the expression of ChM1L mRNA was noted inthe eyeball, the kidney, the stomach, the whole rib and the trachea(FIG. 3( b)). In an adult mouse, ChM1L mRNA was not expressed in thekidney or the stomach, but the ChM1L mRNA was expressed in these tissuesof the fetus suggests that ChM1L is involved in the development andmorphogenesis of these organs. Thus, ChM1L is thought to be alsoassociated with the repair and regeneration of these organs in adults.It was also revealed that ChM1L mRNA is expressed in the trachea. Thus,the ChM1L gene and the ChM1L polypeptide of the present invention,antagonists and agonists to ChM1L including antibody that binds toChM1L, agents that promote or reduce the expression of the ChM1L gene,and the like are considered to be used as therapeutic agents forkidney-related diseases such as chronic kidney failure, stomach-relateddiseases such as gastric cancer and gastric ulcer, and trachea-relatedrespiratory diseases such as chronic bronchitis and asthma.

In the developmental stage of the fetus, the expression of ChM1L mRNA isvery weak at day 10 of gestation, and the expression increases at arounddays 11 to 13 (FIG. 3( c)). On the other hand, though ChM-I, similarlyto ChM1L, increases in expression with the development of the fetus, itwas evidently expressed more strongly than ChM1L on days 10 and 11 ofgestation. It is, therefore, clear that ChM1L lags behind ChM-I in theexpression, and that these molecules have different functions in thedevelopment of the fetus. The increase in ChM1L expression in thedevelopmental stage of the fetus suggests that ChM1L is deeply involvedin the generation of organs and the skeleton. Thus, the ChM1L gene andthe ChM1L polypeptide of the present invention, antagonists and agoniststo ChM1L including antibody that binds to ChM1L, agents that promote orreduce the expression of the ChM1L gene, and the like are considered tobe used as agents for regenerating and repairing organs in the case ofcongenital diseases associated with inadequate development of the organsor acquired damages to the organs. Furthermore, since there aredifferences between ChM1L and ChM-I in the expression in various tissuesin the adult and the fetus, and in the expression during thedevelopmental stage of the fetus, it is expected that these moleculesand drugs that target these molecules, when used as therapeutic agents,have different uses.

Using the sequence of the ChM1L gene of the present invention, it ispossible to produce a polypeptide encoded by said gene by a geneengineering method.

The production of the above polypeptide may be performed by constructinga recombinant DNA that permits the expression of the ChM1L gene of thepresent invention in a host cell, introducing this into a host cell totransform it and then cultivating said transformant.

As the host cell, any of eukaryotic cells and prokaryotic cells can beused.

As the above eukaryotic cells, there may be mentioned vertebrates,yeasts, insect cells, and the like. As the vertebrate cells, there maybe mentioned CHO cells, COS cells, and the like.

As the expression vector for vertebrates, there can be used those thathave a promoter generally located upstream of the gene to be expressed,a polyadenylation site, a transcription termination sequence, and thelike. As the above expression vector, for example there can beillustrated pSV2dhfr (Mol. Cell. Biol., 854, 1981), pcDNA3.1(+)(Invitrogen) and pCAGGS (Gene, 108, 193-200, 1991) that have the SV40early promoter.

As means to express the polypeptide of interest in a eukaryotic cell,there are many systems known per se in the field of art.

For example, as a system that allows expression in yeasts, there can bementioned “Expression of polypeptides in yeast” described in Kokai(Japanese Unexamined Patent Publication) No. 57-159489, as a system thatallows expression in insect cells, there can be mentioned “Process forproducing a recombinant baculovirus expression vector” described inKokai (Japanese Unexamined Patent Publication) No. 60-37988, and as asystem that allows expression in mammalian cells, there can be mentioned“Improvement of eukaryotic expression” described in Kokai (JapaneseUnexamined Patent Publication) No. 2-171198, and there are many others.

The ChM1L gene of the present invention can also be expressed inprokaryotic host cells such as E. coli, Bacillus subtilis, andStreptomyces. For E. coli as above host cell, Escherichia coli strainK12 is often used, and as the vector pBR322 and improved vectors thereofare often used, but they are not limiting and many other known microbialstains and vectors may be used. As the promoter, for example, there canbe mentioned, but not limited to, promoters such as E. coli lactose(lac) and E. coli trp. All of the above promoters have already beencharacterized and are well known to a person skilled in the art, and canbe assembled either synthetically or from known plasmids.

The illustrated DNA sequence of the present invention, plasmids andviruses may have many modifications and variations. For example, due todegeneracy of genetic code, nucleotide substitution can be madethroughout the coding region of a polypeptide. Such a sequence can beeasily deduced from the nucleotide sequence of ChM1L gene of the presentinvention or the amino acid sequence encoded by the gene, and can beassembled by a conventional synthetic method described below. Such asynthetic method may be substantially carried out according to Itakura'smethod (Itakura et al., Science, 198, 1059, 1977) and Crea's method(Crea et al., Proc. Natl. Acad. Sci. USA 75, 5765, 1978). Thus thepresent invention is not limited to the specifically illustratednucleotide sequences, plasmids or viruses.

As methods of introducing the desired recombinant DNA of the presentinvention into a host cell and the following transforming method,various conventional methods may be used. The transformant obtained maybe cultured according to a standard method, from which culture thepolypeptide encoded by the ChM1L gene of the present invention can beproduced. As the culture medium used for this culturing, a commonly usedmedium may be selected as appropriate depending on the host celladopted, and culturing may be carried out under a condition suitable forthe growth of the host cell.

From the foregoing, the above polypeptide can be producedintracellularly, extracellularly, or on the cell membrane of thetransformant. Said polypeptide can be separated and purified utilizing,as desired, the physical properties, chemical properties thereof or thelike by various separation procedures [“Biochemistry Databook II”, pp.1175-1259, First edition, First Print, Jun. 23, 1980, Tokyo KagakuDojin; Biochemistry, 25 (25), 8274-8277 (1986); Eur. J. Biochem., 163,313-321 (1987), and the like]. Specific examples of said methodsinclude, for example, commonly used reconstitution treatment, treatmentwith a polypeptide precipitating agent (salting out), centrifugation,osmotic shock, ultrasonic disruption, ultrafiltration, gel filtration,various chromatographic methods such as adsorption chromatography, ionexchange chromatography, affinity chromatography and high performanceliquid chromatography (HPLC), and combinations thereof. Furthermore, byallowing the expression of the protein in which an affinity tag wasfused to said polypeptide, the tag can be used to perform affinitypurification. As the affinity tag as used herein, there can be mentioneda polyhistidine tag (His tag, Sisk et al., J. Virol., 68, 766, 1994) anda FLAG tag (Hopp et al., Biotechnology 6, 1204-1210, 1988). Theexpression and detection of the ChM1L polypeptide fused to theseaffinity tags can be performed as described in Examples 8 and 9, and itis also possible to purify the ChM1L polypeptide using these tags.

The method of producing the polypeptide encoded by the ChM1L of thepresent invention is outlined as follows, though more detailedexplanations thereof will be made hereinafter in Example 8.

The human and mouse ChM1L gene of the present invention and a geneencoding the ChM1L protein in which a His tag is fused at the C-terminalwere cloned into pcDNA3.1(+) vector (Example 4), which was thentransfected into COS7 cells. About 48 hours later, the culturesupernatant and the cellular components were harvested and weresubjected to a Western blot method in order to detect a ChM1Lrecombinant protein. However, in any of the culture supernatant and thecellular components no expression of ChM1L protein was detected.

Therefore, conditions for detecting the expression of ChM1L recombinantproteins were investigated, and it was found that the use of pCAGGS asthe expression vector enables the detection of expression of saidpolypeptide in COS7 cells. The human and mouse ChM1L gene of the presentinvention and a gene encoding the ChM1L protein in which a His tag isfused at the C-terminal were cloned into pCAGGS vector (Example 4),which was transfected into COS7 cells. About 48 hours later, the culturesupernatant and the cellular components were harvested and weresubjected to a Western blot method in order to detect ChM1L recombinantproteins. No expression of ChM1L recombinant proteins was confirmed inthe culture supernatant, whereas in the cellular components two bandswere detected at around 40 kDa.

Thus, it was revealed that the ChM1L protein is a membrane-boundprotein. On the other hand, it has been confirmed that when ChM-I isexpressed in COS7 cells, it is secreted as a soluble protein in theculture supernatant (Hiraki et al., J. Biol. Chem., 272, 32419-32426,1997). Thus the analysis with COS7 cells revealed that ChM1L and ChM-Iare proteins having different structures. That is, it was shown thatChM1L is a cell membrane-bound protein, while ChM-I is a secretaryprotein, and that the processing mechanisms of these molecules aredifferent. Among the two bands for the ChM1L protein, the band at thehigh molecular weight was found to be a form modified by a N-linkedsugar chain in the Example 10 described below (FIG. 6).

The ChM1L protein thus expressed can affinity purified using aChM1L-specific antibody or an antibody against the tag (His tag) inwhich 6 residues of histidine are fused, a nickel column and the like.

The polypeptide encoded by the ChM1L gene of the present invention maybe any of a membrane-bound polypeptide and a soluble polypeptide havingno property of binding to the cell membrane. For example, there may becases in which after the polypeptide is expressed as a membrane-boundpolypeptide on the cell membrane, it is cleaved to become a solublepolypeptide. Though the ChM1L protein was detected as a membrane-boundprotein in the expression in COS7 cells (Example 8), it may undergoprocessing thereby to be a soluble protein when the host cell or theculture condition is different. Furthermore, the soluble polypeptidethat lacks the transmembrane domain can be expressed by fusing aheterologous signal peptide to the N-terminal.

More specifically, the method of expressing the soluble ChM1L protein isoutlined as follows, though more detailed explanations thereof will bemade hereinafter in Example 9.

A vector was constructed that has integrated, into pCAGGS vector, anucleotide sequence encoding a protein in which the signal sequence ofpreprotrypsin, a FLAG tag, the C-terminal end of the extracellularregion of ChM1L were fused from the N-terminal end (Example 5). TheChM1L protein that was expressed using this vector was secreted into theculture liquid as a soluble protein after the signal sequence ofpreprotrypsin was cleaved (Example 9, FIG. 5).

The soluble ChM1L polypeptide thus secreted into the culture liquid canbe purified using anti-ChM1L antibody or anti-FLAG antibody (Sigma)because a FLAG tag is fused thereto. It is also possible to remove theFLAG tag by cleaving the FLAG fusion protein with enterokinase.

More specifically, the method of purifying the soluble ChM1L protein isoutlined as follows though more detailed explanations thereof will bemade hereinafter in Example 13.

Using a Lipofectamine reagent (GIBCO BRL) according to the instructionattached to the product, pSF-shChM1L was transfected into COS7 cells,and 48 hours later the culture supernatant was harvested. From thisculture supernatant, a soluble ChM1L protein was purified by affinitychromatography using anti-FLAG M2 affinity gel (Sigma) (FIG. 8).

The ChM1L polypeptide of the present invention can be used as apolypeptide-purifying reagent. Said polypeptide bound to a solid supportis very useful for the purification of polypeptides that can bind tosaid peptide by affinity chromatography. As the polypeptide that canbind to the ChM1L polypeptide, there may be illustrated solublepolypeptides, membrane-bound polypeptides, antibodies and the like. Thesoluble ChM1L polypeptide may be readily used for the addition into thecell culture liquid in vitro, or intravenous administration in vivo.

In order to search the activity of the ChM1L polypeptide of the presentinvention, human umbilical vein endothelial cells (HUVECs) were used toanalyze the presence of a angiogenesis-inhibiting activity. Morespecifically, the method is outlined as follows though more detailedexplanations thereof will be made hereinafter in Example 14. When HUVECsare cultured on a plate coated with Matrigel (Becton Dickinson),vascular endothelial cells form a tube-like structure (FIG. 9). When theChM1L polypeptide purified by the above-mentioned affinitychromatography is added to the culture liquid, the formation oftube-like structure of HUVECs was inhibited (FIG. 9). Therefore, it isclear that ChM1L has a angiogenesis-inhibiting activity, and that thesoluble ChM1L polypeptide can be applied as a therapeutic agent fordiseases accompanied by angiogenesis such as diabetic retinopathy,cancer, and rheumatoid arthritis.

Using the polypeptide encoded by the ChM1L gene of the presentinvention, a specific antibody can be generated. An antigen as usedherein includes a polypeptide produced in large quantities according tothe above genetic engineering method or a chemically synthesizedpolypeptide, and an antibody obtained may be any of polyclonal antibodyor monoclonal antibody, and can be effectively used for thepurification, measurement, recognition, and the like of saidpolypeptide. Hence, polyclonal antibodies and monoclonal antibodiesagainst said polypeptide can be used for treatment or the development oftherapeutic methods for diseases that are mediated (directly orindirectly) by said polypeptide, and can also be used as diagnosticreagents for the above diseases.

Antibodies that specifically bind to the polypeptide encoded by theChM1L gene of the present invention can be generated as shown in Example7. That the generated anti-ChM1L polypeptide antibody specifically bindsto said polypeptide was confirmed in the result of Western blot shown inExample 8 (FIG. 4).

Anti-ChM1L polypeptide antibody may also be used for immunostainingtissue sections as described in Example 11. When a rib cartilage wasstained with an anti-ChM1L polypeptide antibody, cells that assume afibroblast-like flat form occurring in such a way as to surround thecartilaginous tissue were specifically stained (FIG. 7). On the otherhand, it has been demonstrated that ChM-I is specifically expressed onchondrocytes and immunostaining has also shown that it is accumulated inthe chondrocytes and matrices other than the chondrocytes (Hiraki etal., J. Biol. Chem., 272, 32429-32426, 1997). It was therefore revealedthat ChM1L and ChM-I are expressed in different cells in the tissueincluding cartilage, which thereby demonstrated that ChM1L and ChM-I aremolecules that have different functions.

The tissue containing the cell group exhibiting a fibroblast-like formthat was demonstrated to be expressing the ChM1L protein byimmunostaining is conventionally termed as perichondrium (Suda et al.,Bone Formation and Bone Absorption and their regulating factors 1, 2,1995). Though there is no definite definition at present on the tissueperichondrium, it is herein intended to mean a tissue containing cellgroups that exhibit a fibroblast-like form.

The cells present in the perichondrium are thought to be a source forchondrocytes that are required for the development of cartilaginoustissues during the process of endochondral bone formation. Hence, theperichondrium is an important tissue that supplies chondrocytes at thetime of skeleton formation during the process of development or bone andcartilage injuries in adults. Furthermore, though the cartilaginoustissue is characterized in that there are no blood vessels, nerves, orlymphatic vessels, the perichondrium is thought to regulate theinfiltration of blood vessels, nerves, or lymphatic vessels into thechondrocytes since the perichondrium is present at the interface ofcartilaginous tissues and other tissues. Thus, though perichondrium isrecognized to be an important tissue, it has no definite definition anddetailed study has not been made at present. One reason for this thatthere are no molecules that are perichondrium-specifically expressedhave been elucidated at all.

Accordingly, if the presence of molecules that are specificallyexpressed in the tissue termed as perichondrium surrounding thecartilaginous tissue is demonstrated, it would provide a very importanttool in the study of perichondrium and cartilaginous tissues.

The ChM1L of the present invention is the only molecule that wasdemonstrated to be perichondrium-specifically expressed, and is thoughtto regulate the infiltration of blood vessels, nerves, or lymphaticvessels into the cartilaginous tissues.

Hence, the discovery of the ChM1L gene and the result of functionalanalysis of ChM1L included herein is believed to provide, from now on, anew perspective on the etiology and the development of therapeuticmethods for diseases in which perichondrium and other ChM1L-expressingtissues including the cartilaginous tissue are involved.

Thus, the ChM1L gene and the ChM1L polypeptide of the present invention,antagonists and agonists to ChM1L including antibody that binds toChM1L, and agents that promote or reduce the expression of the ChM1Lgene are considered to be used as therapeutic agents for diseases inwhich the above ChM1L-expressing cells are involved.

The above expression analysis of mRNA and the result of immunostainingrevealed that the ChM1L gene and the polypeptide encoded thereby of thepresent invention are expressed in the brain, the eyeball, the skeletalmuscle, the thyroid, the whole rib including cartilage, the kidney, thestomach, the trachea, and cells that assume a fibroblast-like flat formoccurring in such a way as to surround the cartilaginous tissue. Itsuggests, therefore, that the ChM1L gene and the polypeptide encodedthereby of the present invention may be involved in diseases associatedwith the above tissues that have been confirmed to express them, such asdiabetic retinopathy, muscular dystrophy, Basedow's disease, chronickidney failure, stomach cancer, chronic bronchitis, osteoarthritis andrheumatoid arthritis.

Hence, the ChM1L gene and the ChM1L polypeptide of the presentinvention, antagonists and agonists to ChM1L including antibody thatbinds to ChM1L, and agents that promote or reduce the expression of theChM1L gene are considered to be used as therapeutic agents for thesediseases.

EXAMPLES

The present invention will now be explained more specifically withreference to the following examples. It should be noted, however, thatthe present invention is not limited to these examples.

Example 1 Analysis of the ChM1L Amino Acid Sequence

The homology of amino acid residues of ChM-I and ChM1L was compared(FIG. 1( a)). The amino acid sequence was represented by onealphabetical letter. ChM1L has a homology with ChM-I throughout themolecule, but it was found that ChM1L has a particularly high homologywith the C-terminal of ChM-I that is extracellularly secreted followingthe processing of ChM-I.

The homology of amino acid sequences of human, mouse and rat ChM1L wascompared (FIG. 1( b)). The ChM1L polypeptide is composed of 317 aminoacids in humans, mice, and rats, but the 300 amino acid residues wereidentical in the three (about 95%).

The degree of hydrophobicity of ChM-I and ChM1L is shown in the figure(FIG. 2). In both of ChM-I and ChM1L, a large hydrophobic peak isobserved in the N-terminal end. This hydrophobic region ischaracteristically observed in the cell membrane-bound proteins, and itwas demonstrated that both of ChM1L and ChM-I are type II membrane-boundproteins.

Example 2 Cloning of the ChM1L Gene

Using the amino acid sequence (Genbank accession number M16441) of humanChM-I, TBLASTN search was performed for the Expressed sequence tag database (dbEST) in the DNA data bank of Japan (DDBJ). As a result, an ESTfile, Genbank accession number AI123839 was detected as a novel genefragment having a homology with ChM-I.

Using Clontech's Human fetus Marathon-Ready ™ cDNA, cDNA was amplifiedby the RACE method according to the instruction attached to the product.Primers were synthesized from the nucleotide sequence obtained from theabove dbEST, and ExTaq polymerase (Takara Shuzo) was used according tothe according to the instruction attached to the product. Using GeneAmp™PCR System 9700 (PE Applied Biosystems), PCR reaction was carried outfor 30 cycles with each cycle comprising 96° C. for 30 seconds, 60° C.for 30 seconds, and 72° C. for one minute, and finally incubated at 72°C. for 6 minutes to obtain a PCR reaction mixture. One tenth of thereaction mixture was added, and the second PCR was performed in the samecondition.

The PCR product obtained was subjected to electrophoresis on a 1%agarose gel containing ethidium bromide and then the gel was observedunder UV irradiation to examine DNA bands. The amplified fragments wereexcised from the gel, and were purified using QIAquick Gel ExtractionKit (QIAGEN) according to the instruction attached to the product.

The nucleotide sequence of the purified fragment was determined using aDNA sequencer (ABI PRISM™ 310 Genetic Analyzer) of PE Applied Biosystemsand ABI PRISM ™ BigDye Terminator Cycle Sequencing Ready Reaction kit.

The nucleotide sequence of human ChM1L cDNA is shown in SEQ ID NO: 1 andits amino acid sequence is shown in SEQ ID NO: 2.

Since the amino acid sequence encoded by the human ChM1L generepresented by SEQ ID NO: 1 has a homology with human ChM-I, the genewas decided to be termed as the ChM1L gene (ChM-I like gene).

The coding sequence (CDS) of Human ChM1L cDNA was amplified by PCR,electrophoresed on agarose, and then was purified, which was then clonedusing pCR-Script™ Amp cloning kit (Stratagene) according to theinstruction attached to the product. The sequence of the primers used inPCR are shown in SEQ ID NO: 7 (forward primer) and SEQ ID NO: 8 (reverseprimer). The ChM1L gene sequence that has been integrated into thevector was determined using ABI PRISM™ 310 Genetic Analyzer of PEApplied Biosystems and ABI PRISM™ BigDye Terminator Cycle SequencingReady Reaction kit.

Using the amino acid sequence (SEQ ID NO: 2) of human ChM1L, TBLASTNsearch was carried out as described in the above human case. As aresult, as a gene fragment that encodes mouse ChM1L, EST file, Genbankaccession number AV009191 was detected, and as a gene fragment thatencodes rat ChM1L, EST file, Genbank accession number AI112003 wasdetected. Using Mouse 11-day Embryo Marathon-Ready™ cDNA and RatSkeletal muscle Marathon-Ready™ cDNA by Clontech, sequences of mouse andrat ChM1L genes were determined by the RACE method as described in theisolation of the human ChM1L gene.

The nucleotide sequence of mouse ChM1L cDNA is shown in SEQ ID NO: 3 andthe amino acid sequence is shown in SEQ ID NO: 4. The nucleotidesequence of rat ChM1L cDNA is shown in SEQ ID NO: 5 and the amino acidsequence is shown in SEQ ID NO: 6.

The coding sequences (CDS) of mouse and rat ChM1L cDNA were amplified byPCR, and were purified after agarose electrophoresis, which were thencloned using pCR-Script™ Amp cloning kit (Stratagene) according to theinstruction attached to the product. The sequences of the primers usedin PCR of the mouse gene are shown in SEQ ID NO: 9 (forward primer) andSEQ ID NO: 10 (reverse primer). The sequences of the primers used in PCRof the rat gene are shown in SEQ ID NO: 11 (forward primer) and SEQ IDNO: 12 (reverse primer). The sequence of the ChM1L gene that wasintegrated into the vector was determined using ABI PRISM™ 310 GeneticAnalyzer of PE Applied Biosystems and ABI PRISM™ BigDye Terminator CycleSequencing Ready Reaction kit.

The names of human, mouse and rat ChM1L genes constructed in thisExample are abbreviated to:

the vector containing the human ChM1L gene: pCR-hChM1L

the vector containing the mouse ChM1L gene: pCR-mChM1L

the vector containing the rat ChM1L gene: pCR-rChM1L

Example 3 Construction of Vectors Containing Genes Encoding Human andMouse ChM1L Proteins in which 6 Residues of Histidine are Fused to theC-Terminal

The coding sequences (CDS) of human and mouse ChM1L cDNA were amplifiedby PCR, and were purified after agarose electrophoresis, which were thencloned using pCR-Script SK(+) vector that has been improved so that 6histidine residues (His tag) may be fused to the C-terminal andpCR-Script™ Amp cloning kit (Stratagene) according to the instructionattached to the product. The sequence of the primers used in PCR of thehuman gene are shown in SEQ ID NO: 7 (forward primer) and SEQ ID NO: 13(reverse primer). The sequence of the primers used in PCR of the mousegene are shown in SEQ ID NO: 9 (forward primer) and SEQ ID NO: 14(reverse primer). That a nucleotide sequence encoding a protein in whichHis tags are fused to the C-terminal of ChM1L was determined using ABIPRISM™ 310 Genetic Analyzer (PE Applied Biosystems) and ABI PRISM™BigDye Terminator Cycle Sequencing Ready Reaction kit according to theinstruction attached to the product. The amino acid sequences of humanand mouse ChM1L in which His tags were fused to the C-terminal are shownin SEQ ID NO: 17 and 18, and the nucleotide sequence encoding them areshown in SEQ ID NO: 15 and 16.

The genes encoding the ChM1L protein constructed in this Example inwhich a His tag was fused are abbreviated to:

the vector containing a gene encoding the protein in which human ChM1Land His tags were fused: pCR-hChM1LHis

the vector containing a gene encoding the protein in which mouse ChM1Land His tags were fused: pCR-mChM1LHis

Example 4 Construction of Expression Vectors

In order to express the ChM1L gene in a mammalian cell, CDSs wereexcised from the above pCR-hChM1L, pCR-mChM1L, pCR-hChM1LHis, andpCR-mChM1LHis with restriction enzymes EcoRI and NotI, wereelectrophoresed on agarose, and then the desired bands were purified,which were ligated to pcDNA3.1(+) (Invitrogen) and pCAGGS (Gene, 108,193-200, 1991) using Ligation high (Toyobo) according to the instructionattached to the product. The solutions after the ligation reaction weresubjected to transformation using E. coli JM109 competent cells (TakaraShuzo) according to the instruction attached to the product. Afterpurifying the plasmids, the integration of the desired gene wasconfirmed by restriction enzyme reactions and agarose electrophoresis.

The vectors constructed in this Example are abbreviated to:

pcDNA3.1(+) vectors containing the hChM1L, mChM1L, hChM1LHis andmChM1LHis: pcDNA-hChM1L, pcDNA-mChM1L, pcDNA-hChM1LHis, andpcDNA-mChM1LHis

pCAGGS vectors containing the hChM1L, mChM1L, hChM1LHis and mChM1LHis:pCAGGS-hChM1L, pCAGGS-mChM1L, pCAGGS-hChM1LHis, and pCAGGS-mChM1LHis

Example 5 Construction of Vectors that Express Human Soluble ChM1LProtein to which a FLAG Tag is Fused

The FLAG tag (Sigma) as used herein is a hydrophilic marker peptidecomprising eight amino acids (Asp Tyr Lys Asp Asp Asp Asp Lys), and thelast five amino acids (Asp Asp Asp Asp Lys) is a recognition sequencefor enterokinase. The vectors constructed in this Example can express aprotein in which the signal sequence of preprotrypsin, a FLAG tag, theC-terminal end of the extracellular region of ChM1L were fused from theN-terminal end. The protein that was expressed using this vector issecreted into the culture liquid as a soluble protein after the signalsequence of preprotrypsin is cleaved, as explained in detail hereinafterin Example 9. Since a FLAG tag is fused to the protein expressed withthis vector, the protein can be purified using anti-FLAG antibody(Sigma) and by cleaving the fusion protein with enterokinase, the FLAGtag can also be removed.

A vector was constructed in which a nucleotide sequence (SEQ ID NO: 19,contained in pFLAG-CMV-1 vector manufactured by Sigma) encoding thesignal sequence of preprotrypsin and a FLAG tag (SEQ ID NO: 20) from theN-terminal was integrated into the pCAGGS vector (hereinafter referredto as pSF vector). A nucleotide sequence (nucleotide sequence No. 684 to1020 of SEQ ID NO: 1) encoding the amino acids No. 212 to 317 of humanChM1L represented by SEQ ID NO: 2 and the translation termination codonwas amplified by the PCR method, and the amplified product wasintegrated into the 3′-end of the nucleotide sequence encoding the FLAGtag of the pSF vector. The sequences of primers used for PCR are shownin SEQ ID NO: 21 (forward primer) and SEQ ID NO: 8 (reverse primer). Theintegration of the sequence of interest into the constructed vector wasconfirmed using a ABI PRISM™ 310 Genetic Analyzer (PE AppliedBiosystems) and ABI PRISM™ BigDye Terminator Cycle Sequencing ReadyReaction kit according to the instruction attached to the product. Thenucleotide sequence of this Example integrated into the vector is shownin SEQ ID NO: 22, and the amino acid sequence encoded thereby is shownin SEQ ID NO: 23. The vector constructed in this Example will beabbreviated to pSF-shChM1L.

Example 6 Expression Analysis of ChM1L mRNA Expression Analysis of ChM1LmRNA in Various Tissues of an Adult (10-Week Old): FIG. 3(a)

A 10-week old C57BL/6 mouse was dissected and each tissue was extracted,which was immediately frozen in liquid nitrogen. The frozen tissue wasground into small pieces, and using IOSGEN (Nippon Gene) according tothe instruction attached to the product the total RNA of each tissue wasobtained. With one μg of total RNA of each tissue as template, 20 μl ofcDNA was synthesized using Superscript II preamplification kit (GIBCOBRL) according to the instruction attached to the product. In RT-PCR,the total liquid volume of the reaction system was set at 50 μl, and foreach tissue 0.5 μl of cDNA, 0.25 μl of ExTaq polymerase (Takara Shuzo)were used, to which the forward primer (SEQ ID NO: 9) and the reverseprimer (SEQ ID NO: 10) were added to a concentration of 0.2 μm. UsingGeneAmp™ PCR System 9700 (PE Applied Biosystems), PCR amplification wasperformed for 30 cycles with each cycle comprising 96° C. for 30seconds, 60° C. for 30 seconds, and 72° C. for one minute. The reactionmixture obtained was subjected to electrophoresis on a 1% agarose gelcontaining ethidium bromide and then the gel was observed under UVirradiation to examine the expression of ChM1L mRNA in each tissue.

As shown in FIG. 3( a), the expression of ChM1L mRNA in each tissue ofan adult mouse was observed in the brain, the eyeball, the skeletalmuscle, the whole rib, and the thyroid. The expression of ChM-I in micehas been confirmed in the eyeball, the thymus, the cartilage, and thewhole rib. It is therefore clear that ChM1L and ChM-I are expressed indifferent tissues in the living body, suggesting that theirphysiological functions are different.

Expression Analysis of ChM1L mRNA in Various Tissues of a Fetus (Day 17of Gestation): FIG. 3(b)

A fetus of C57BL/6 mouse on day 17 of gestation was removed by Caesareansection. Each tissue was taken out, and was immediately frozen in liquidnitrogen. The extraction of total RNA from the frozen tissue, cDNAsynthesis, and RT-PCR were performed as described in the above<Expression analysis of ChM1L mRNA in various tissues of an adultmouse>.

As shown in FIG. 3( b), the expression of ChM1L mRNA in each tissue of afetus mouse was observed in the eyeball, the kidney, the stomach, thewhole rib, and the trachea. In the fetus, expression in the kidney andthe stomach was observed, in which no expression was observed in theadult mouse. It is therefore likely that ChM1L is involved in thedevelopment and morphogenesis of these organs, and is also considered tobe involved in the repair and regeneration of organs. It was alsorevealed that ChM1L mRNA is expressed in the trachea.

Expression Analysis of ChM1L mRNA during the Developmental Stage of aFetus: FIG. 3(c)

A fetus of C57BL/6 mouse on each day from day 10 of gestation tochildbirth was removed by Caesarean section. Each the whole fetuses wasfrozen in liquid nitrogen. The extraction of total RNA from the frozenfetus, cDNA synthesis, and the implementation of RT-PCR were performedas described in the above <Expression analysis of ChM1L mRNA in varioustissues of an adult mouse>.

The analysis of ChM-I mRNA was carried out using a fusion protein (SEQID NO: 23) and a reverse primer (SEQ ID NO: 24) under the same conditionas above.

As shown in FIG. 3( c), the expression of ChM1L mRNA during thedevelopmental stage of the fetus is very weak on day 10 of gestation andis increased in expression on days 11 to 13. On the other hand, thoughthe expression of ChM-I was also increased as for ChM1L, it exhibited anevidently stronger expression than ChM1L on days 10 and 11 of gestation.It is therefore clear that the expression of ChM1L lags behind ChM-Iduring the developmental stage of the fetus, and that these moleculeshave different functions in the fetus development.

Example 7 Generation of Anti-ChM1L Peptide Polyclonal Antibody

A peptide having cysteine at the C-terminal of the sequence from 245 to252 residues shown in SEQ ID NO: 2 of human ChM1L was chemicallysynthesized. To this synthetic peptide, MBS/KLH(m-maleimidobenzoyl-N-hydroxysuccinimide ester/keyhole limpethemocyanin, Boehringer Mannheim) was coupled. After the complex wasdissolved in physiological saline, an equal amount of Freund's completeadjuvant (FCA) was added, which was sonicated to prepare an emulsion.This emulsion was subcutaneously given to a rabbit as the initialimmunization. Four weeks after the initial immunization, a boosterimmunization was carried out using Freund's incomplete adjuvant (FIA) tothe femoral muscle, and thereafter immunization by subcutaneousadministration was carried out for four times at an interval of abouttwo weeks or four weeks. During the booster immunization, blood waspartially taken from the auricle, and after the final immunization theentire blood was taken and serum was separated. By affinity purificationusing a peptide column, an anti-ChM1L peptide polyclonal antibody wasobtained.

Example 8 Analysis of Human and Mouse ChM1L Recombinant Protein byWestern Blotting: FIG. 4

Using the lipofectamine reagent (GIBCO BRL) according to the instructionattached to the product, pCAGGS, pCAGGS-hChM1L and pCAGGS-mChM1L (FIGS.4( a) and (c)), or pCAGGS, pCAGGS-hChM1LHis and pCAGGS-mChM1LHis (FIGS.4( b) and (d)) were transfected into COS7 cells. About 48 hours afterthe transfection, the culture supernatant and the cellular componentswere subjected to sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) on a 12.5% gel, and then transferred to anitrocellulose membrane. A primary antibody reaction and a secondaryantibody reaction were carried out, and then subjected to a colordeveloping reaction using the ECLplus reagent (Amersham PharmaciaBiotech) according to the instruction attached to the product. In theWestern blot in which pCAGGS, pCAGGS-hChM1L and pCAGGS-mChM1L weretransfected, anti-ChM1L polyclonal antibody described in the aboveExample was used as the primary antibody and horseradish peroxidase(HRP)-labelled anti-rabbit IgG antibody (DAKO) was used as the secondaryantibody, and in the Western blot in which pCAGGS, pCAGGS-hChM1LHis andpCAGGS-mChM1LHis were transfected, anti-His tag antibody (Invitrogen)was used as the primary antibody and HRP-labelled anti-mouse IgGantibody (Amersham Pharmacia Biotech) was used as the secondaryantibody.

SDS-PAGE was carried out for the same samples as in Western blot, andthe results of staining with Coomassie brilliant blue (CBB) are shown inFIGS. 4( a) and (b).

As a result of Western blot, no ChM1L band was confirmed in any of theculture supernatants. In cellular components, as shown in FIGS. 4( b)and (d), recombinant ChM1L protein was detected as two bands at around40 kDa whether anti-ChM1L peptide antibody or anti-His tag antibody wasused. As will be described in detail in the Examples below, the band atthe high molecular weight was confirmed to be a form in which a N-linkedglycosylation.

Example 9 Analysis of Soluble Human ChM1L Recombinant Protein by aWestern Blot Method: FIG. 5

Using the lipofectamine reagent (GIBCO BRL) according to the instructionattached to the product, pCAGGS and pSF-shChM1L were transfected intoCOS7 cells. After the culture supernatants were subjected to SDS-PAGE ona 12.5% gel, they were transferred to a nitrocellulose membrane.Anti-FLAG M2 antibody (Sigma) was used as the primary antibody andHRP-labelled anti-mouse IgG antibody (DAKO) was used as the secondaryantibody, and the ECLplus reagent (Amersham Pharmacia Biotech) was usedaccording to the instruction attached to the product to perform colordevelopment reaction.

As shown in FIG. 5, soluble human ChM1L protein was detected as a singleband at around 17-18 kDa.

Example 10 Deglycosylation Analysis of ChM1L Recombinant Protein

Using the lipofectamine reagent (GIBCO BRL) according to the instructionattached to the product, pCAGGS-mChM1LHis was transfected into COS7cells. After PBS saline containing 2% SDS was added to a dish, cellswere harvested by a scraper, and the suspension was heated at 95° C. for60 minutes. The supernatant was treated with SDS-OUT™ SDS Precipitationkit (Pierce) to remove SDS. Using the protein solution thus obtained, adeglycosylation was carried out using the Enzymatic Deglycosylation Kit(BIO RAD) according to the instruction attached to the product to treatthe above protein solution with NANase II, O-glycosidase DS and PNGaseF. After the reaction mixture was subjected to SDS-PAGE on a 12.5% gel,it was transferred to a nitrocellulose membrane. Anti-His tag antibody(Invitrogen) was used as the primary antibody and HRP-labelledanti-mouse IgG antibody (Amersham Pharmacia Biotech) was used as thesecondary antibody, and the ECLplus reagent (Amersham Pharmacia Biotech)was used according to the instruction attached to the product to performcolor development reaction.

As shown in FIG. 6, the band of ChM1L protein at the high molecularweight disappeared only when treated with pNGase F (lanes 2 and 5). Itwas therefore demonstrated that the ChM1L protein has been modified witha N-linked sugar chain.

Example 11 Analysis of ChM1L Protein at Cartilago Costalis byImmunostaining

An about 10-week old C57BL/6 mouse was dissected to remove the wholerib, which was fixed in a 10 mM phosphate buffer (pH 7.4) (PBS)containing 4% paraformaldehyde, embedded in paraffin, and then sectionswere prepared. Each step of immunostaining was carried out usingHistfine SAB-PO® kit (Nichirei) according to the instruction attached tothe product, of which outline is as follows: After deparaffinization,endogenous peroxidase was digested with a 3% hydrogen peroxide. Afterwashing with PBS followed by blocking with 10% normal goat serum, theabove-mentioned anti-ChM1L peptide antibody at a dilution of 1/160 wasadded and incubated overnight at 4° C. As a negative control, rabbit IgGwas used. After biotin-labelled anti-rabbit IgG antibody andperoxidase-labelled streptoavidin were allowed to react,3,3-diaminobendizine/4HCl was added to perform a color developmentreaction. The nucleus was stained with haematoxylin, enclosed, and thenobserved.

As shown in FIG. 7, ChM1L protein is expressed in cells that assume afibroblast-like flat form occurring in such a way as to surround thecartilaginous tissue. On the other hand, there were no expressionsobserved in the cartilage cell in which the expression of ChM-I has beenreported.

Example 12 Chromosome Mapping of the Human ChM1L Gene

Using the gene sequence (SEQ ID NO: 1) of human ChM1L, BLASTN search wasperformed for the entire DDBJ data from the DNA data bank of Japan(DDBJ). As a result, Genbank accession number AL035608 was detected asthe genome sequence of the ChM1L gene. AL035608 is a sequence mapped onchromosome X. It is therefore clear that the human ChM1L gene is presenton chromosome X.

Example 13 Purification of a Soluble Human ChM1L Recombinant Protein

Using a Lipofectamine reagent (GIBCO BRL) according to the instructionattached to the product, pSF-shChM1L was transfected into COS7 cells,and 48 hours later the culture supernatant was harvested. Usinganti-FLAG M2 affinity gel (Sigma), an affinity column was prepared, andthe culture supernatant was applied to the column. After washing thecolumn three times in 25 mM Tris-HCl, 150 mM NaCl (pH 7.4), it waseluted with 0.1 M glycine-HCl (pH 3.5), and the eluent was neutralizedwith a 1/20 volume of 1M Tris-HCl (pH 9.5).

The culture supernatant and the eluent were subjected to SDA-PAGE andthen were stained with Coomassie brilliant blue (CBB), the result ofwhich is shown in FIG. 8. Though there are a variety of proteins in theculture supernatant (FIG. 8, lane 1), soluble human ChM1L protein wasconfirmed as an about 20 kDa band in the eluent. This revealed thatsoluble human ChM1L protein was concentrated and purified by the aboveprocedure (FIG. 8, lane 2).

Example 14 Study on the Effect of Inhibiting Angiogenesis using HumanUmbilical Vein Endothelial Cells

Human umbilical vein endothelial cells (HUVECs, Clonetics) were culturedin a exclusive medium (EGM™-2 Bullet Kit™, Clonetics) for endothelialcells. To a 12-well plate, Growth factor reduced Matrigel (BectonDickinson) was added to 600 μl/well, which was then incubated at 37° C.for 30 minutes. Using a heparin-free exclusive medium for endothelialcells diluted ⅛ in the essential medium (EBM™-2, Clonetics) forendothelial cells, a cell suspension containing 5×10⁴ cells/ml of HUVECswere prepared.

Each test substance solution was prepared as a solution in which a 1/20volume of 1M Tris-HCl (pH 9.5) was added to 0.1M glycine-HCl (pH 3.5),and 200 μl/well of it was treated. The above buffer and bovine serumalbumin (BSA) at 20 μg/well as the negative control, platelet factor 4(PF-4, CHEMICON) at 1 and 10 μg/well as the positive control, and theeluted fractions of Example 13 at 10 and 20 μg/well as the soluble humanChM1L recombinant protein were treated. Two ml of the cell suspension(1×10⁵ cells) and 200 μl of the test substance solution were mixed, andseeded into a 12-well plate coated with Growth factor reduced Matrigel.Nine hours later, the formation of tube-like structures was examined andwere a photograph was taken. The result is shown in FIG. 9. In thenegative control, HUVECs formed tube-like structures (FIGS. 9( a) and(b)), but when ChM1L at 20 μg/well (FIG. 9( d)) was treated theformation of tube-like structures was inhibited as compared to thenegative control.

It was therefore revealed that ChM1L has an effect of inhibitingangiogenesis and thus the soluble ChM1L polypeptide can be used as atherapeutic agent for disease accompanied by angiogenesis such asdiabetic retinopathy, cancer, and rheumatoid arthritis.

1. A monoclonal antibody or an antigen-binding fragment thereof whichspecifically binds to a protein consisting of amino acids 202 to 317 ofSEQ ID NO:
 2. 2. An isolated polyclonal antibody or an antigen-bindingfragment thereof which specifically binds to a protein consisting ofamino acids 202 to 317 of SEQ ID NO:
 2. 3. A hybridoma producing amonoclonal antibody or an antigen-binding fragment thereof, obtainableby cell fusion of a myeloma cell and an antibody-producing cellimmunized with a protein consisting of amino acids 202 to 317 of SEQ IDNO:
 2. 4. A kit comprising (1) a protein consisting of amino acids 202to 317 of SEQ ID NO: 2, and (2) a monoclonal antibody or antigen-bindingfragment thereof, and/or a polyclonal antibody or antigen-bindingfragment thereof, which specifically binds to said protein.
 5. Acomposition comprising a monoclonal antibody or antigen-binding fragmentthereof, or a polyclonal antibody or antigen-binding fragment thereof,which specifically binds to a protein consisting of amino acids 202 to317 of SEQ ID NO: 2, and at least one carrier.
 6. A compositioncomprising a monoclonal antibody or antigen-binding fragment thereof, ora polyclonal antibody or antigen-binding fragment thereof, whichspecifically binds to a protein consisting of amino acids 202 to 317 ofSEQ ID NO: 2, and at least one carrier.